Process for the preparation of cellulose film, cellulose film produced thereby, artificial skin graft and its use

ABSTRACT

There is described a process for the preparation of cellulose film, comprising the steps of preparing a culture medium having as nutrients sources of nitrogen and of carbohydrate, seeding this medium with a culture of acetobacter, species Xylinium; incubating the culture at temperatures which permit bacterial activity for a time suitable to the final intended use of the film, and removing the formed film from the culture medium for dehydration in a distended state. The film thus prepared is suitable for use as an artificial skin graft, a separating membrane, or artificial leather.

The present invention relates to artificial skin grafts, and moreparticularly provides a process for the preparation of artificial skin.

Skin lesions have always been a grave problem to be confronted bymedicine, particularly those lesions caused by burns. In these cases,the patient suffers extensive hydro-electrolitic losses, these lossesbeing greater as the extent of the burned area increases. There is acritical period of approximately 36 hours in which substantial loss ofblood fluids occurs in the burned region. After this period, this fluidloss is interrupted by the closure of the tissues, and the oppositeeffect is noted, i.e. a fluid retention which causes swelling. In caseswhere the area affected is great, the consequences can be fatal.

These extreme symptoms provoked by loss of skin through burns from heatradiation or chemical products also occur to a lesser extent in caseswhere skin loss is occasioned by abrasion or other mechanical causes.The conventional treatment, which is long and painful, consists in thecontinued application of medications, such as ointment, lubricatedgauze, and bandages for retaining the gauze in place. The affectedregion is kept covered, and the patient receives large doses ofantibiotics to prevent infection. For many years the above describedproblems have been tackled by applying skin substitutes to the affectedarea. The incessant search for solutions to these problems isillustrated by a vast bibliography of more than 120 studies published inthe last ten years throughout the world, concentrating on attempt tofind a skin substitute having the same properties and characteristics ashuman skin.

To date, the following human skin substitutes exist:

1. Autogenous grafting, in which skin is taken from another location onthe patients body;

2. Heterogeneous grafting, which may be of two types:

firstly, either human skin from donors or cadavers, or amniotic membranefrom donors may be used;

secondly, skin from pigs or bovine embryos, or artificial skins such assilicon, collagen, mixtures of collagen and silicon, orpolytetrafluoroethylenepolyurethane.

Briefly, these items may be described in the following manner:

Autogenous grafting

This involves a surgical operation, extracting an area of the patientsown skin from an elected donor region, to be immediately applied overthe lesioned area. Since this causes a trauma equivalent to a seconddegree burn, this is only justifiable in patients who have third degreeburns.

Heterogenous grafting

The first method uses human skin from donors, this method beingextremely rarely used since as well as requiring donors to submitthemselves to extensive traumas, the benefits of this type of graftingare transitory as the graft only lasts for a maximum of two weeks.

Human skin from cadavres

This type of grafting is almost never used, due to the inherentdifficulties in obtaining cadavres at the appropriate time, togetherwith their entire medical histories. There also exist ethical and legaldifficulties, as well as objections from the family of the deceased.

Amniotic membrane

This requires a careful choice of donor, and the total absence of riskof contamination. The membrane must be prepared by a specialized team ina medical center, and must be done within twelve hours. As to itsdurability, like artificial skin substitutes it does not last more thana week.

Pigskin

Among the various animal skins exhaustively texted for use as human skinsubstitutes, pigskin presented the best results due to its anatomiccharacteristics similar to those of man. However, the necessaryprecausions and the preparation methods give this skin anatomiccharacteristics similar to man. However, the necessary care in thechoice of animal and the preparation of a piece of skin make this optionextremely laborious and complex.

The piglet of less than 5 months old, must be absolutely healthy andmust have a skin free of any injury. The piglet is electrocuted and thendecapitated so that all the blood is lost. A specialized team, workingin a sterilizable atmosphere similar to a medical center removes all theskin of the piglet in a first stage, and thereafter prepares it intosheets using electrical equipment. The sheets must be stored underrefrigeration (maximum temperature 4° C.) in physiological serum withantibiotics. This "skin" once applied to the patient has a limiteddurability of two weeks.

Artificial skins

Various studies have been carried out over the years in attempts to usesynthetic products as skin substitutes for lesions where skin loss hasoccurred. Two lines of research are worthy of note, the first being thesearch for a substitute prepared totally from synthetic substances suchas silicon, polyurethane, and others, the second attempting to associatea synthetic film of organic material prepared from derivatives of animalblood such as for example collagen.

The first, a completely synthetic skin, has not yet passed through theinvestigative stage and no product has been placed on the market. Thesecond refers to an artificial skin, prepared from two integral layers.The layer which enters in contact with the lesion is formed from driedorganic material, preferably derived from animal bood, and the other issilicon, polyurethane, or the like and functions as a support, thislayer being exposed. The organic part is absorbed by the organism, andthe support is then rejected. A product is being marketed, and itscharacteristics are described in Brazilian patent No. PI 7800285,patentee Battelle Memorial Institute.

The present invention resolves the problems described above in asuprising and totally unexpected way, by providing a cellulosic film ofvarying thickness. The film is translucent, and has selectivepermeability. The film is pleasant to the touch, and very similar tohuman skin, the film being semi-permeable, elastic, and having greatadhesive power when in contact with the exudate which covers the lesionarea. The film has none of the previously mentioned disadvantages, andin addition provides the following advantages in relation to the priorart:

The film is of low cost, easy preparation, and may be sterilized withoutdeterioration.

The esthetic aspect is pleasant, which favours the patientpsychologically.

The film is easily applied, the use of dressings being unnecessary.

The film is adapted to any body location, since it easily adheres tolesioned regions by their exudate, and has sufficient elasticity topermit movement.

The film is easily stored, since it is not perishable and does not needrefrigeration.

Application of the film immediately relieves pain since it protects thenerve endings.

The strong adhesion of the film to the wound reduces the proliferationof germs, and leaves the wound hermetically sealed.

Hydro-electrolytic losses are reduced. A shortening of the scar formingtime in second degree burns, and the tissue granulation in third degreeburns.

No formation of retractile scarring occurs.

Since there is no need to change dressings, the risk of contamination isdeminished.

There is no allergic reaction.

The film permits visual inspection of the development of the treatment,by its translucence. This is in contrast with the artificial skinspresently existent.

The film must be applied delicately over the injured region, so as toprevent the inclusion of air bubbles or bood secretions between thewound and the film. Once in position on the exudate, and covering thelesion completely, the film gradually and slowly absorbes the exudate.Due to this slowness, the film provides conditions for coagulation ofthe exudate within the flm, thus forming a bridge between the film andthe lesioned area. Also due to the slow absorption, coagulation occursbefore the exudate has penetrated the membrane, coagulation occurring intwo areas;

(a) the microscopic spaces existing between the films fibers;

(b) the surface of the injury.

From this, there results a perfect adhesion preventinghydro-electrolytic losses and substantially reducing the risk ofinfection. Additionally, since the nerve endings are isolated, the painof the injury is immediately reduced.

The film, therefore, makes it possible for the patient to reconstitutethe epithelial tissues, since the lesioned area is isolated andcontamination is avoided. Under these conditions, there is no necessityto change dressings, since the adhesion of the film to the lesion willonly cease gradually, as the tissue regenerates. The film is formedtotally from cellulose, an inert substance, and does not have anymedicinal action on the lesioned zone. After having provided theorganism with conditions to regenerate the lesioned tissues, the filmceases to adhere to the patient, and leaves no residue whatsoever on theregenerated area. The film becomes unstuck, with exactly the samecharacteristics it had at the moment of its application, and may beregarded simply as a prosthesis or temporary implant.

Since the cellulose film is strong and inert, it permits of practicallyall types of sterilization. It may be stored at any temperature, anddoes not require special conditions. It is of unlimited durability. Thefilm has determined permeability to liquids and air, a characteristicmolecular weight and structure, a predictable thickness when dehydrated,in addition to other specific physical characteristics.

The following physical and spectroscopic studies were made with theobjective of better defining the material of the present invention.

To this end, a sample of the film was partially whitened using a 30%sodium hypochloride solution, the whitened part being subsequently dyed,leaving a remainder in its original, non-whitened condition. Thefollowing results were obtained:

    ______________________________________                                                       Film                                                           Tests            Whitened    Non-Whitened                                     ______________________________________                                        1. Permeability to air,                                                                        11          8                                                Bendtsen, mL/min                                                              (150 WG)                                                                      2. Resistance to air, Gurley,                                                                  more than   more than                                        s/100 mL                                                                      NBR 7152/82      1800 seconds                                                                              1800 seconds                                     (ABNT MB-1271/79)                                                             3. Tensile strength, kg/cm.sup.2                                                               --          7,92                                             NBR 7462/82                                                                   (ABNT-MB 57/68)                                                               4. Elongation, % --          74                                               NBR 7462/82                                                                   (ABNT-MB 57/68)                                                               ______________________________________                                         Note:                                                                         For the physical tests, the film was brought to a moisture content of 25      ± 5% and the proof bodies were packed and tested at a relative humidit     of 65 ± 2% and a temperature of 20 ± 1° C.                  

Spectroscopic analysis revealed that both the whitened and non-whitenedfilms were of cellulosic nature.

In the spectroscopic study, the following reference table was used, thetable having been taken from "infra-red spectra of cellulose and itsderivatives" by Rostislav Georgievich Zhbankov, and published in 1966.

                  REFERENCE TABLE                                                 ______________________________________                                        Group/bonding                                                                              Compound     Wave number cm.sup.-1                               ______________________________________                                         ##STR1##    cellulose and its derivatives                                                              from 1400 to 1200                                    ##STR2##    cellulose and its derivatives                                                              from 1400 to 1300                                    ##STR3##    cellulose and its derivatives                                                              from 1200 to 1100 from 1150 to                      ______________________________________                                                                  1000                                            

The process for preparation of the film is a biosynthesis which takesplace during the reproduction of bacteria of the order pseudomonadales,from the family of pseudononadaceas, of the genus acetobacter, speciesXylinum. These bacteria, seeded in a culture medium, having carbohydratenutrients associated with a nitrogen source, produce a cellulosiccapsular zoogloea which surrounds the microorganisms.

This zoogloea has a cartilaginous appearance and a thickness whichvaries between 0.2 and 400 mm. After seeding the bacteria, aschizogenous vegetative reproduction process is initiated; the cell isdivided in two equal parts and grows until a central ring is formed,where the bacterium divides. In the case of acetobacter, after cellulardivision the new bacteria is aggregated to its originating bacteria andimmediately initiates its own reproductive process. Under idealconditions, a further division occurs each 20 minutes.

The acetobacters are surrounded by a cellulosic capsule and afterdivision they remain agglomerated, forming chains. This characteristic,allied to the speed of reproduction, propiciates the formation of acellulosic film by the linking of the bacterial chains with theirrespective capsules.

The thickness of the film is variable and depends on various factors,such as the dosage of nutrients, temperature, time, saturation point ofthe colony, and the type of culture medium.

The zoogloea of the acetobacters is a fabric formed by the interweavingof the bacterial chains surrounded by their cellulosic capsules.Proceeding with the dehydration of the zoogloea, there are obtainedsheets of pure cellulose which, by their characteristics, permit theiruse as raw material for various industrial purposes, among thesedermatological prostheses, suture medium, or leather substitutes.

After dehydration the microscopic spaces existing between the fibres ofthe membrane offer selective permeability characteristics.

The thickness of the film is a function of the following variables,interalia:

(a) the content of carbohydrates in the culture medium;

(b) the content of nitrogen in the culture medium;

(c) the temperature;

(d) the duration of the period of formation of the zoogloea.

Various examples will now be given to illustrate the invention, theexamples being non-limitative.

EXAMPLE 1

A culture medium was prepared from an infusion of 20 g of Tea Sinensisin 10 liters of water. The infusion was filtered, and 1 kg of sugar wasadded to the filtrate, which was then mixed.

To the culture medium obtained, 10 ml of a culture of acetobacter,species Xylinum was added, and was incubated at 28° C. for a period of36 hours, a thin film being formed of approximately 0.2 mm thickness.

The film is removed from the medium, and is optionally boiled dependingon its intended use and is then dehydrated at ambient temperature onsupports, in a distended state. The film thus obtained is then ready foruse as artificial skin or leather. The culture medium separated from theskin is filtered, and is made up with the nutrient solution initiallyused to compensate for losses which occur during the biosynthesisprocess.

The temperature of incubation and the concentration of carbohydrates inthe culture medium are not critical, and may vary between very lowlevels which still permit bacterial activity and high levels which stillpermit existence of the film without causing deformation thereof.

The temperature of dehydration is also not critical, and normal heatsources may be used.

EXAMPLE 2

The process of example 1 is followed, the culture being left to incubateduring a period of 96 hours, whereupon a film of zoogloea of 3 mmthickness was obtained, the film being usable after dehydration as anartificial skin graft.

EXAMPLE 3

The process of example 1 was used, using as a source of nitrogen, mate.

A film identical to that of example 1 was obtained, with the samepredehydration thickness, the incubation time being 72 hours.

EXAMPLE 4

A film prepared in accordance with the previous examples was taken anddelicately applied on a lesioned region where loss of epithelial tissuehad occurred, so that between the film and the injury no air bubbles orblood secretions were formed. Once the film was applied over theexudate, completely covering the lesion, the film absorbed the exudatebut did not permit loss of exudate nor entry of air.

The film therefore forms a new skin over the tissue, mechanicallyeliminating pain symptoms by isolating the nerve ending. The lesionregenerates after a time, and the film spontaneously lifts from theregion after its regeneration leaving visible a new epithelial tissuecompletely regenerated.

The cellulose film of the invention may also be used as a surgicalsuture, given the necessary preparation, and presents a high knotstrength.

The film may also be used as a separating membrane, as artificialleather, for tennis racket strings, or any other of the uses to whichcellulose is normally put.

I claim:
 1. A liquid gas permeable cellulose film, produced according tothe process comprising the steps of:preparing a culture medium having asnutrients a carbohydrate nutrient and a source of nitrogen; seeding inthis medium a culture of Acetobacter, species xylinum; incubating theculture at temperatures which permit the activity of the bacteria duringa time suitable for the intended use of the film; and withdrawing theformed film from the culture medium and dehydrating it while it isstretched.
 2. A cellulose film according to claim 1 wherein the nitrogensource is Tea Sinensis, and the carbohydrate nutrient is sucrose.
 3. Acellulose film according to claim 1 having a thickness of 0.2 to 3 mmprior to dehydration.
 4. A cellulose film according to claim 2 having athickness of 0.2 to 3 mm prior to dehydration.
 5. An artificial skingraft comprising a dehydrated liquid and gas permeable cellulose filmaccording to claim 1 which has been cut to size after the dehydrationstep, sterilized, and packaged.
 6. An artificial skin graft comprising adehydrated liquid and gas permeable cellulose film according to claim 2which has been cut to size after the dehydration step, sterilized, andpackaged.
 7. An artificial skin graft comprising a dehydrated liquid andgas permeable cellulose film according to claim 3 which has been cut tosize after the dehydration step, sterilized, and packaged.
 8. Anartificial skin graft comprising a liquid and gas permeable cellulosefilm according to claim
 1. 9. An artificial skin graft comprising aliquid and gas permeable cellulose film according to claim
 2. 10. Anartificial skin graft comprising a liquid and gas permeable cellulosefilm according to claim 3.